Electrophoresis through agarose or polyacrylamide gels is the standard method used to separate, identify, quantify, and purify nucleic acid fragments. The technique is simple, rapid to perform, and capable of resolving fragments of nucleic acids that cannot be separated adequately by other procedures. Typically, the location of nucleic acids within the gel can be determined by staining with low concentrations of the fluorescent intercalating dye ethidium bromide; bands containing as little as 1-10 ng of nucleic acid can be detected by direct examination of the gel in ultraviolet light. If necessary, these bands of nucleic acids can be recovered from the gel and used for a variety of purposes, including, for example, cloning.
Agarose gels are cast by melting agarose in the presence of a desired buffer until a clear transparent solution is obtained. The melted solution is then poured into a mold and allowed to harden. Upon hardening the agarose forms a matrix. When an electric field is applied across the gel, nucleic acids, which are negatively charged at neutral pH, migrate toward the anode. To obtain maximum resolution of nucleic acid fragments greater than 2 kb in size, agarose gel voltage is typically no more than 5 V/cm. The rate of migration is determined by a number of parameters, including the molecular size of the nucleic acid strands. Larger molecules migrate more slowly because of greater frictional drag and because they work their way through the pores of the gel less efficiently than smaller molecules.
Horizontal slab gels are usually poured onto a glass plate or plastic tray that can be installed on a platform in an electrophoresis tank. The position of stained nucleic acid strands within the gel are measured by photographing the gel while it is illuminated with ultraviolet light. Most typically, the gel image is recorded onto film, although digital imaging systems that are sensitive to light at ˜300 nm wavelengths can also be used. Using a long exposure time and a strong UV light source, fluorescence emitted by a little as 1 ng of nucleic acids can be recorded on film.
Polyacrylamide gels are almost always poured between two glass plates that are held apart by spacers (0.5-2.0 mm) and sealed with electrical tape. In this arrangement, most of the acrylamide solution is shielded from exposure to the air, so that inhibition of polymerization by oxygen is confined to a narrow layer at the top of the gel. Polyacrylimide gels have advantages of greater resolving power, greater molecular capacity, and higher purity extraction of recovered nucleic acids compared to agarose gels.
Like nucleic acid electrophoresis, separation of proteins or peptides within a gel can be accomplished by the application of an electrical potential that results in a rate of molecular migration that is dependent on the charge of the molecule, rather than its mass. The electrophoretic mobility of a molecule is equal to the net charge on the molecule divided by its frictional coefficient.
The gel is a loosely cross-linked network that functions to stabilize the protein. For proteins, gels composed of polysaccharide agarose or polyacrylamide are typically used. The percentage of gel used is gauged according to the size of the proteins being separated. For the finest separations, gradient gels are made with a continuous increase in gel percentage along the length of the slab. This approach leads to optimum separation of components in a mixture and the sharpest protein solvent boundaries. At the completion of the run, a dye that stains the proteins is added to the gel to establish the locations of protein bands.
Another electrophoretic method frequently used for characterizing proteins is based on differences in their isoelectric points—called isoelectric focusing. The apparatus consists of a narrow tube containing a gel and a mixture of ampholytes, which are small molecules with positive and negative charges. The ampholytes have a wide range of isoelectric points, and are allowed to distribute in the column under the influence of an electric field. This step creates a pH gradient from one end of the gel to the other, as each particular ampholyte comes to rest at a position coincident with its isoelectric point. At this stage, a solution of proteins is introduced into the gel. The proteins migrate in the electric field until each reaches a point in which the pH resulting from the ampholyte gradient exactly equals its own isoelectric point. Isoelectric focusing thus provides a way of both accurately determining a protein's isoelectric point and effecting separations among proteins whose isoelectric points can differ by as little as a few hundredths of a pH unit.
A popular method for protein molecular weight estimation is called sodium dodecyl sulfate (SDS) gel electrophoresis. This procedure uses the same types of polyacrylamide gels as described previously. The mixture of proteins to be characterized is first completely denatured by the addition of SDS (a detergent) and mercaptoethanol, followed by a brief heating step. The resulting unfolded polypeptide chains have relatively large numbers of SDS molecules bound to them. Because each bound SDS molecule contributes two negative charges, the “native” protein charge is masked by the bound SDS—and thus the net charge of the denatured molecule is proportional to its molecular weight.
An extension of the electrophoretic method combines isoelectric focusing with SDS gel electrophoresis to produce a two-dimensional electrophoretogram. This technique is most valuable for the analysis of very complex protein mixtures. First the sample is run in a one-dimensional pH gradient gel (isoelectric focusing). The resulting narrow strip of gel, containing a partially separated mixture of proteins, is placed alongside a square slab of SDS gel. An electric field is imposed so that the sample moves at a right angle to its motion in the first gel.
Two-dimensional electrophoresis (2-DE) is the highest resolution analytical method for proteins available (S.D. Patterson, “Proteomics: the industrialization of protein chemistry,” Current Opinion in Biotechnology, Vol. 11, p. 413-418, 2000). The main use of the technology is as a protein profiling expression tool. Complex protein mixtures from paired (or multiple) samples are separated to allow comparison of either their relative abundance using image analysis tools. Another advantage of the method is the ability to extract proteins (or peptides derived from it) from the gel matrix for subsequent identification and/or characterization.
These methods of electrophoresis have several disadvantages. Staining reagent toxicity is one disadvantage of conventional electrophoresis methodologies. For example, ethidium bromide is a powerful mutagen, and is moderately toxic. All solutions containing it must be decontaminated before disposal. Typically, decontamination is performed by diluting solutions with water, mixing with KMnO4, mixing with HCl, and NaOH. While the procedure can reduce the mutagenic activity of ethidium bromide by 3000-fold, the procedure takes several hours and significant residual mutagenic activity has been reported in some cases. In addition, the KMnO4 reagent is itself an irritant and explosive. Thus, the procedure must be carried out carefully by trained staff within a chemical fume hood. Alternative decontamination procedures have been reported, each with various levels of effectiveness, cost, time, and safety.
Another disadvantage of conventional electrophoresis techniques is poor resolution sensitivity. For example, two-dimensional gel electrophoresis can have poor resolution. Out of the entire complement of the genome of about 100,000 genes, a given cell line may express about 10,000 genes—and an even higher number is expressed in tissues. Furthermore, the dynamic range of abundance of proteins in biological samples can be as high as 106. Because even the best two-dimensional gels can routinely resolve no more than 1,000 proteins, only the most abundant proteins can be visualized by gel electrophoresis. (A. Pandey and M. Mann, Nature, Vol. 405, p. 837-846, 2000). Additionally, known methods of electrophoresis modify the separated molecules due to label addition and removal. Other methods require the use of special UV-sensitive film.
Therefore, new electrophoretic techniques that do not use mutagenic materials, that provide high resolution sensitivity, that do not modify the separated molecules, and that do not require specialized film are needed in the art.